I seek to define structure/function relationships of cation-chloride cotransporters, with particular focus on the kidney-specific absorptive Na+,K+,2CI- cotransporter (NKCC2). This ion transport protein facilitates the reabsorption of Na+ and Cl- in the mammalian kidney, thus playing a major role in body electrolyte and water homeostasis. The specific goals of this proposal are: 1) to define structure/function relationships in a region of NKCC2 hypothesized to be involved in ion translocation, and 2) to test the hypothesis that mutations in NKCC2, linked to altered blood pressure homeostasis in humans, have significant effects on the ion transport function of this protein. To accomplish these goals, mutant NKCC2s will be engineered using site-directed mutagenesis followed by functional characterization using a Xenopus laevis oocyte expression system. The research proposed here is critical to the identification and understanding of mechanisms underlying alterations in renal salt and water reabsorption, the diagnosis of human disease, and the targeting of therapeutic intervention. Relevance: In humans, the kidney functions to reabsorb salt and water from the body before it is lost via urination, and this task is carried out, in part, by an ion transport protein at the focus of this investigation. In some individuals this protein does not function properly leading to diseases that result in loss of normal kidney function. In order to be able to diagnose and treat these diseases, we must understand the structure (what it looks like) and function (how it works) of this ion transport protein.